Project Summary: During mitotic cell division (mitosis), replicated chromosomes must be accurately segregated into two daughter cells to ensure normal development and growth in all eukaryotes. Errors in chromosome segregation can lead to aneuploidy, a hallmark of cancer and a host of associated health problems. As a result, a major frontier in biomedical research is to understand the mechanisms that govern the assembly and maintenance of the mitotic spindle, a microtubule-based bipolar machine responsible for accurate chromosome segregation during mitosis. It is well established that proper assembly and maintenance of a bipolar mitotic spindle requires the coordinated action of many microtubule-based kinesin motors (mitotic kinesins). However, the mechanisms of individual mitotic kinesins and their regulation by partner proteins and coordination during spindle assembly and maintenance remain poorly understood. The long-term goal of the PI is to reconstitute bipolar mitotic spindles in vitro from purified proteins and to fully dissect the mechanisms, regulation and coordination of mitotic kinesins in spindle assembly and maintenance. The focus of this project is on mitotic kinesin-14s, which are known to form an antagonistic pair with kinesin-5s to drive bipolar spindle assembly. The PI has established several foundational findings for this project, including that (1) the mitotic kinesin-14 KlpA from Aspergillus nidulans (and its ortholog in Aspergillus niger) exhibits processive plus-end-directed motility on single microtubules, and (2) the same KlpA in complex with two well-conserved partner proteins switches to processive minus-end-directed motility on single microtubules. The latter discovery represents a first set of in vitro studies showing that a mitotic kinesin-14 depends on partner proteins to gain processive minus-end-directed motility. Building on these findings, this project is aimed at filling two major open questions that are key to understanding how kinesin-14 opposes kinesin-5 in bipolar spindle assembly: (1) What is the molecular basis underlying processive plus-end-directed kinesin-14 motility on single microtubules? (2) How are kinesin-14s regulated by partner proteins to drive bipolar spindle assembly? This project uses an innovative combination of multiple techniques, including single-molecule total internal reflection fluorescence microscopy, dark field nanoparticle tracking, two-color high-precision fluorescence tracking, genetic incorporation of unnatural amino acids, and high-resolution optical trapping. Results from this project will not only markedly broaden current understanding of kinesin motility mechanisms but also provide a mechanistic understanding of kinesin-14 regulation in bipolar spindle assembly. Furthermore, this work will be a stepping stone toward the long-term goal of a complete understanding of the mechanisms, regulation and coordination of kinesins in mitotic spindle assembly and maintenance, and also provide new insights into how mitotic kinesins and their partner proteins can be targeted for therapeutic interventions.